Infection with Mycobacterium tuberculosis (Mtb) results in chronic inflammation of the lung and other tissues, which is difficult to treat with current tuberculosis (TB) drugs. The challenge to effectively curing patients with active TB is due to the persistence of drug-tolerant bacilli sequestered in granulomas with caseous necrosis or cavitation. Existing treatment regimes that are often complicated by toxic side effects consist of months or years of combination antimicrobial drugs, which are deemed necessary to effectively eradicate drug-tolerant bacilli. We propose a novel TB treatment strategy, targeting drug-tolerant Mtb using a class of small molecular weight, anti-biofilm compounds to be combined with conventional TB drugs. This strategy could significantly improve the efficacy of current TB drug therapy by more rapidly eradicating, persistent, drug-tolerant bacilli. Our in vitr and in vivo data show that Mtb forms extracellular, biofilm-like microbial communities as a strategy to survive TB drug therapy. From studying the Guinea pig TB model we determined that these drug-tolerant bacilli adopt the biofilm-like mode of existence when associated with lysed or necrotic leukocytes. To mimic this unique in vivo microenvironment we developed a novel in vitro assay in which extracellular, biofilm-like communities of Mtb are cultured on an attachment matrix derived from lysed human leukocytes. Similar to what is seen in vivo, our data show that Mtb expresses extreme in vitro drug tolerance, which is reversed by anti-biofilm drugs, rendering bacilli again susceptible to TB drugs. Our data show that these novel compounds disperse or inhibit the formation of biofilms formed by a variety of bacteria including M. smegmatis and increases the susceptibility of Mtb to isoniazid. The effectiveness of this strategy will be further tested using our in vitro Mtb drug tolerance assay to show 1) anti-biofilm compounds disperse and inhibit the formation of Mtb biofilm-like communities in vitro and 2) that combining anti-biofilm compounds with TB drugs effectively eradicates drug-tolerant Mtb associated with host-derived macromolecules. We will then determine whether combining anti-biofilm and TB drugs in the Guinea pig Mtb infection model is more effective at eliminating drug- tolerant Mtb than TB or biofilm drugs alone. In a series of mechanistic studies, we will use biotinylated, anti- biofilm compounds as molecular probes to identify Mtb specific virulence factors unique to the drug-tolerant phenotype. By determining the identity of Mtb adhesins or protein intermediates of metabolic pathways unique to drug-tolerant Mtb, additional compounds will be designed, synthesized and tested for biological activity. Successful completion of these highly innovative, translational studies will establish the feasibility of using anti- biofilm compounds as adjunct therapy to treat chronic Mtb in conjunction with existing TB drugs. We will also determine if anti-biofilm compounds combined with TB drugs are more effective at targeting drug-tolerant bacilli compared to TB drugs alone. Finally, we will reveal the mechanisms of action of our current anti-biofilm compounds and the identity of other druggable targets unique to drug-tolerant Mtb.